Regio- and Stereospecificity of Filipin Hydroxylation

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Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from .... We previously reported the crystal structures of CYP105P1 in three different ...

Supplemental Material can be found at: http://www.jbc.org/content/suppl/2010/04/07/M109.092460.DC1.html THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 22, pp. 16844 –16853, May 28, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis*□ S

Received for publication, December 7, 2009, and in revised form, March 27, 2010 Published, JBC Papers in Press, April 7, 2010, DOI 10.1074/jbc.M109.092460

Lian-Hua Xu‡, Shinya Fushinobu‡, Satoshi Takamatsu§, Takayoshi Wakagi‡, Haruo Ikeda§, and Hirofumi Shoun‡1 From the ‡Department of Biotechnology, Graduate School of Agriculture and Life Sciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 and the §Kitasato Institute for Life Sciences, Kitasato University, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan

Macrolide compounds have toxic effects on a wide variety of organisms including pathogens, and therefore, their clinical use as antibiotics has been widely developed (1). Postpolyketide modifications of macrolides by cytochrome P450 (P450 or CYP)2 hydroxylases provide molecular diversity to

* This work was supported by Grants-in-aid for Scientific Research 20248009 (to H. S.) and 20310122 (to H. I.) from the Japan Society for the Promotion of Science. The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1, Figs. S1–S4, and a movie. The atomic coordinates and structure factors (codes 3ABA and 3ABB) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). 1 To whom correspondence should be addressed. Tel./Fax: 81-3-5841-5148; E-mail: [email protected] 2 The abbreviations used are: P450 or CYP, cytochrome P450; HPLC, high performance liquid chromatography; r.m.s.d., root-mean square deviations; MES, 2-morpholinoethanesulfonic acid. □ S

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these macrolides during their biosynthesis (2, 3). P450s are hemoproteins whose fifth axial heme iron ligand is a thiolate group found in a variety of organisms (4, 5). A majority of P450s catalyze monooxygenation (hydroxylation or epoxidation) of hydrophobic substrates (6) using a dioxygen bound as the sixth iron ligand as well as various redox systems responsible for the cleavage of the O-O bond (7–9). Understanding the molecular mechanisms of P450 enzymes during the biosynthesis of natural products would facilitate their potential uses in producing new drugs (10). The crystal structures of macrolide monooxygenases complexed with their substrates or analogues have been determined for P450eryF (CYP107A1; erythromycin biosynthesis) (11, 12), P450 EryK (CYP113A1; erythromycin biosynthesis) (13), P450 PikC (CYP107L1; narbomycin and pikromycin biosynthesis) (14), and P450epoK (CYP167A1; epothilone biosynthesis) (12) (see supplemental Fig. S1). The 28-membered polyene macrolide antibiotic filipin is widely used as a probe for cholesterol in biological membranes (15, 16) and a prominent diagnostic tool for type C Niemann-Pick disease (17, 18). Filipin, originally isolated from Streptomyces filipinensis as a filipin complex (19), is composed of four components (see Fig. 1) (20). The major component (53%) is filipin III, and its stereochemical configuration has been determined (21, 22). Filipin I (4%) lacks two hydroxyl groups of filipin III located at positions C1⬘ and C26 (23). Filipin II (25%) is 1⬘-deoxyfilipin III (24). Filipin IV (18%) is isomeric to filipin III and is probably epimeric at C1⬘ or C3 (25). A solution NMR study has shown that the large 28-membered ring is rigid, stabilized by both intramolecular hydrogen bonds of syn 1,3-polyols and a conjugated pentaene moiety, whereas the lateral aliphatic chain is highly flexible (26). A gene cluster for filipin biosynthesis was recently identified in the genome of Streptomyces avermitilis (27). The gene cluster contains two P450 genes, CYP105P1 (PteC, SAV413) and CYP105D6 (PteD, SAV412) as well as genes encoding modular polyketide synthases (pteA1-pteA5), ferredoxin (fdxI, pteE), and putative zinc binding dehydrogenase (pteB). The filipin biosynthetic gene cluster is regulated by the negative regulator aveI along with the biosynthetic genes for other antibiotics including avermectin and oligomycin (28). Analysis of P450 gene deletion mutants revealed that CYP105P1 and CYP105D6 catalyze hydroxylations at posiVOLUME 285 • NUMBER 22 • MAY 28, 2010

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The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C NiemannPick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the “filipin complex.” CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1ⴕ, respectively. Here, we determined the crystal structure of the CYP105P1filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a Bⴕ helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes.

Crystal Structures of Filipin Hydroxylases TABLE 1 Data collection and refinement statistics Data set Data collection statistics Beam line Wavelength (Å) Space group Unit cell (Å) Resolution (Å)a Total reflections Unique reflections Completeness (%)a Redundancya Mean I/␴(I) a Rmerge (%)a Refinement statistics Protein Data Bank code Resolution range (Å) No. of reflections R-factor/ Rfree (%) No. of atoms TLS groups (residue no.) Average B-factor (Å2) Protein Heme Filipin I Water SO42⫺ r.m.s.d. from ideal values Bond lengths (Å) Bond angles (degrees) Ramachandran Plot (%)b Favored Allowed Outlier

EXPERIMENTAL PROCEDURES Protein Preparation and Spectroscopy—CYP105P1 protein was expressed and purified as described previously (29). The primers used to amplify the CYP105D6 gene were 5⬘-CCC ATA TGA CTG AGA CCG AAA TCC GCC TC-3⬘ and 5⬘-GGA CTA GTT CAG TGG TGG TGG TGC CAG ACG ACG GGG AGC TCG ATC-3⬘ (bold type and underlined sequences represent the restriction endonuclease sites and His4 tag, respectively). The expression plasmid was constructed using pET-17b (Novagen, Madison, WI). CYP105D6 protein was expressed in Escherichia coli C43 (DE3) cultured in Terrific Broth medium containing 12 g/liter Bacto-tryptone, 24 g/liter yeast extract, 8 g/liter glycerol, 17 mM KH2PO4, 72 mM K2HPO4, and 100 mg/l ampicillin at 25 °C for 24 h. After the correction of the cells by centrifugation, cells were suspended in 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 10 mM imidazole, 0.1 mM dithiothreitol, and 10% (v/v) glycerol. Cell extracts were obtained by sonication and followed by centrifugation to remove cell debris. A fraction containing the protein was purified on a HiTrap Chelating HP 5-ml column (GE Healthcare) with a linear gradient of 10 –500 mM imidazole. After dialysis, the protein was further purified on a Resource Q column (GE Healthcare) with a linear gradient of 0 – 0.5 M NaCl. The final step of purification was on a Superdex 200 column (GE Healthcare) separated with 10 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 0.1 mM dithiothreitol, and 10% (v/v) glycerol. The purified enzyme appeared as a single band corresponding to a molecular mass of 44 kDa on SDS-PAGE (data not shown). The absorbance ratio of proteins purified in this manner was greater than 2.0 at 420 nm as compared with that at 280 nm. The P450 content measured by CO difference spectroscopy was also checked to verify purification quality. Purification of Filipin I—Spores of S. avermitilis strain ⌬pteC/pteD were inoculated into a medium containing 5 g/liter glucose, 15 g/liter soy flour, and 5 g/liter yeast extract (pH 7.0) and cultured with agitation at 30 °C for 48 h. A portion of the culture (1% seed) was inoculated into filipin production medium containing 20 g/liter dextrin, 2 g/liter glucose, 15 g/li3

៮ mura, unpublished data. H. Ikeda, M. Doi, A. Arisawa, Y. Fujii, and S. O

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a b

CYP105P1-Filipin I complex

CYP105D6 Ligand-free

PF-BL5A 1.000 P41212 a ⫽ b ⫽ 91.368 c ⫽ 151.239 50.00-1.80 (1.86-1.80) 829,838 59,975 99.9 (100.0) 13.8 (13.9) 38.7 (3.2) 8.8 (46.5)

PF-AR NW12A 1.000 P3121 a ⫽ b ⫽ 67.533 c ⫽ 182.089 50.00-2.30 (2.38-2.30) 238,132 22,279 100.0 (100.0) 10.7 (9.8) 25.3 (4.1) 9.8 (46.0)

3ABA 39.74–1.80 55,867 18.8/23.8 3775 7–82, 83–146, 147–323, 324–403

3ABB 33.77–2.30 21,082 16.0/22.1 3274 11–92, 93–192, 193–408

18.3 21.4 25.9 30.8 46.1 0.028 2.138 98.7 1.0 0.3

23.2 16.6 30.6 0.022 2.016 96.8 2.9 0.3

Values in parentheses are for the highest resolution shell. Determined by RAMPAGE server (49).

ter soy flour, 3 g/liter yeast extract, and 3 g/liter CaCO3 (pH 7.0) and cultured at 30 °C for 5 days on a rotary shaker. Mycelia from the culture medium (3 liters) were collected using a Bu¨chner funnel and extracted twice with acetone. The sample was concentrated using a rotary evaporator, transferred to a separating funnel, and extracted twice with ethyl acetate. Solid anhydrous sodium sulfate was added for dehydration and then concentrated to dryness. The sample was dissolved with chloroform and then placed into a silica gel column (60 ⫻ 400 mm) eluted with chloroform/methanol (1:0, 10:1, and 4:1). The eluate was collected in 15-ml fractions. The retention time of a yellow band containing filipin I was about 30 min in chloroform/methanol (4:1). The sample was concentrated to dryness, dissolved with methanol, and filtered. The compound was finally purified by preparative HPLC (Pegasil-ODS 20 ⫻ 250 mm, Senshu Scientific Co., Tokyo, Japan) eluted with acetonitrile/methanol/ water 55:20:25 at 340 nm of UV detection at a 9 ml/min flow rate. The separated compound was concentrated by evaporation and extracted twice with ethyl acetate. The extract was concentrated to dryness using an evaporator and dried in a vacuum desiccator. Spectroscopy—UV-visible absorption spectra measurements and titration experiments were performed essentially using the same methods as described previously (29). For the titrations of filipin I to CYP105P1, 1 ml of assay buffer containing 50 mM potassium phosphate (pH 7.5), 0.1 mM dithiothreitol, 0.1 mM EDTA, and 10% (v/v) glycerol was used. The protein concentration was 5.1 ␮M, and 2 mM filipin I stock solution was added. A nonlinear fitting with a quadratic equation was applied to determine the Kd using Kaleidagraph (Synergy, Reading, PA): JOURNAL OF BIOLOGICAL CHEMISTRY

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tions C26 and C1⬘, respectively.3 The amino acid sequence identity between CYP105P1 and CYP105D6 is 36.7% when the whole lengths of both sequences are used for alignment. We previously reported the crystal structures of CYP105P1 in three different states (29). The ligand-free wild-type structure provides a unique state in which the His-72 residue in the BC loop is ligated to the heme iron atom. When compared with the 4-phenylimidazole-bound wild-type and ligand-free H72A mutant structures, it is suggested that the high flexibility of the BC loop of this enzyme is a key feature for incorporating the large hydrophobic filipin substrate. In this report we present two crystal structures of the filipin hydroxylases: the structures of CYP105P1-filipin I complex and ligand-free CYP105D6. Our present study provides a concrete structural basis for filipin hydroxylation at position C26 and an insight into the different substrate specificities of these similar P450 enzymes, both belonging to the CYP105 family.

Crystal Structures of Filipin Hydroxylases

Downloaded from www.jbc.org at KITASATO-DAIGAKU-IGAKUBU, on December 19, 2012 FIGURE 1. Analytical HPLC of the reaction products from filipin I by CYP105P1 and CYP105D6. A, control reaction without P450 enzymes is shown. B, reaction with CYP105P1 is shown. C, shown is the reaction with CYP105D6 on the sample in panel B. D, reaction with CYP105D6 is shown. E, reaction with CYP105P1 on the sample in panel D is shown. Structures of filipin I, filipin II (1⬘-deoxyfilipin III), and filipin III are also shown. The configurations of the stereogenic centers in filipin III are 1⬘R, 2R, 3S, 5S, 7S, 9R, 11R, 13R, 15S, 26S, and 27R.

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Crystal Structures of Filipin Hydroxylases

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FIGURE 2. Spectral changes of CYP105P1 (ferric resting state) upon the addition of increasing concentrations of filipin I (A), its difference spectra (B), and the titration curve calculated using the values of absorption differences at 387 and 422 nm (C).

shown in Table 1. Figures were prepared using PyMol (DeLano Scientific LLC, Palo Alto, CA).

RESULTS Spectral Characterizations and Measurements of Filipin Hydroxylase Activities—Recombinant proteins of CYP105P1 and CYP105D6 with His4 tag at the C termini were expressed in E. coli cells and purified to homogeneity. Spectral characterization of purified CYP105P1 has been described previously (29). UV-visible absorption spectra of purified CYP105D6 in the ferric (resting), dithionite-reduced, and dithionite-reduced plus CO states are shown in supplemental Fig. S2. These spectra show that the protein was folded properly. To examine the substrate specificities of CYP105P1 and CYP105D6 in vitro, the purified enzymes and filipin I were incubated with the electron transport system of spinach ferredoxin and reductase (Fig. 1). Filipin I was produced and purified JOURNAL OF BIOLOGICAL CHEMISTRY

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⌬A ⫽ (Bmax/2E){(Kd ⫹ E ⫹ L) ⫺ {(Kd ⫹ E ⫹ L)2 ⫺ 4E/L}1/2}, where Bmax is the maximum absorbance difference extrapolated to infinite ligand enzyme concentration, L is the ligand concentration, and E is the total enzyme concentration. Measurement of Filipin Hydroxylase Activity—The reaction mixture (200 ␮l) contained 50 mM potassium phosphate (pH 7.5), 0.1 mM EDTA, 0.1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM NADPH, 0.05 units of spinach ferredoxin:NADP⫹ reductase (Sigma), 0.015 mg of spinach ferredoxin (Sigma), 0.2 mM filipin I, and 1 ␮M P450 enzyme. The reaction was started by adding NADPH, and the mixture was incubated at 30 °C for 90 min. The reaction was terminated by mixing with 1.5-fold volume of ethyl acetate. The mixture was centrifuged at 6000 ⫻ g for 1 min to separate phases, and a portion of the ethyl acetate layer was concentrated to dryness using a centrifugal evaporator. The sample was dissolved with methanol and subjected to analytical HPLC (Pegasil-ODS 4.6 ⫻ 250 mm, Senshu Scientific Co.) at a 0.8 ml/min flow rate. The peak position of each compound was determined according to a previous study; the structure of each compound was analyzed by fast atom bombardment mass spectrometry, 1H NMR and 13C NMR.3 Filipin III, filipin II, 1⬘-hydroxyfilipin I, and filipin I eluted at 4.6, 7.2, 11.3, and 21.2 min, respectively. Crystallography—For crystallization, protein was concentrated to ⬎20 mg/ml in 10 mM Tris-HCl (pH 7.5), 0.5 M NaCl, and 0.1 mM EDTA (protein solution buffer). Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA). After three mixing cycles with filipin I and concentration by the centrifugal filter device, three more mixing cycles with the protein solution buffer and concentration were performed to remove unbound filipin I from the solution. Crystallization was performed using the sitting drop vapor diffusion method. CYP105P1 crystals complexed with filipin I were grown at 25 °C by mixing 1 ␮l of the protein solution (10 mg/ml protein) and 1 ␮l of the reservoir solution containing 2.0 M (NH4)2SO4, 0.2 M Li2SO4, and 0.1 M N-cyclohexyl-3-aminopropanesulfonic acid (pH 10.5). CYP105D6 crystals were grown at 25 °C by mixing 1 ␮l of the protein solution (8 mg/ml protein) and 1 ␮l of the reservoir solution containing 4.0 M sodium formate (pH 8.0). X-ray diffraction data were collected at the BL-5A and NW12A stations at the Photon Factory, High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. After cryoprotection with 20% (v/v) glycerol, crystals were flash-cooled in a nitrogen stream at 100 K. Diffraction images were processed using the HKL2000 program suite (30). The initial phases were determined by molecular replacement using MOLREP (31). The ligand-free CYP105P1 structure was used as a search model. Manual model rebuilding, introduction of water molecules, and refinement were performed using Coot (32) and Refmac5 (33). The topology and parameter file for filipin I was generated based on the solution NMR structure of filipin III (26) using the PRODRG server (34). In the final refinement stage, bulk solvent correction and TLS (parameterization of the translation, libration, and screw rotation displacements of pseudorigid bodies) refinement with the groups defined by the TLSMD server (35) was applied. Data collection and refinement statistics are

Crystal Structures of Filipin Hydroxylases

FIGURE 3. Spectral changes of CYP105D6 (ferric resting state) upon the addition of increasing concentrations of filipin I (A), its difference spectra (B), and the titration curve calculated using the values of absorption differences at 387 and 420 nm (C). A nonlinear fitting with a quadratic equation was applied to the titration curve.

from a mutant S. avermitilis strain in which both CYP105P1 and CYP105D6 genes were deleted (⌬pteC/pteD).3 After incubation with CYP105P1, 50.2% of filipin I was converted to filipin II (Fig. 1B). The reaction product was extracted by ethyl acetate and then incubated with CYP105D6 (Fig. 1C). The second reaction resulted in 43.0% conversion to filipin III, and 15.8, 24.4, and 16.8% of filipin II, 1⬘-hydroxyfilipin, and filipin I were detected. Therefore, in the second reaction catalyzed by CYP105D6, 73.1% of filipin II and 69.2% of filipin I were converted to filipin III and 1⬘-hydroxyfilipin I, respectively. After incubation with CYP105D6, 34.6% of filipin I was converted to 1⬘-hydroxyfilipin I (Fig. 1D). Subsequent reaction with CYP105P1 resulted in 27.5% conversion to filipin III and 47.5,

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7.6, and 17.4% of filipin II, 1⬘-hydroxyfilipin, and filipin I were detected. Therefore, in the second reaction catalyzed by CYP105P1, 57.9% of 1⬘-hydroxyfilipin I and 73.2% of filipin I were converted to filipin III and filipin II, respectively. These results indicated that these enzymes hydroxylate filipin I at different positions. The activity against filipin I was higher for CYP105P1 than CYP105D6. In addition to filipin I, CYP105P1 and CYP105D6 can hydroxylate 1⬘-hydroxyfilipin I and filipin II, respectively. CYP105D6 showed preference to filipin II over filipin I, and CYP105P1 showed preference to filipin I over 1⬘-hydroxyfilipin I. Figs. 2 and 3 show spectral titration results of CYP105P1 and CYP105D6 with filipin I, respectively. The spectra of CYP105P1 illustrate a typical type I spectral shift of the Soret peak (419 nm) to 391 nm. The spectral change of CYP105D6 was relatively smaller, but the difference spectrum clearly shows a type I shift. Filipin I has three absorption maxima around the 320 –360-nm region and a shoulder at 305 nm due to vibrational progression of a polyene (19). These peaks exhibited perturbations on binding to CYP105P1 and CYP105D6, and positive peaks were observed in the difference spectra at 329 –330, 346 –347, and 362–368 nm. The titration curve of CYP105P1 indicated strong binding to the ligand with a stoichiometry of about 1:1 ⬃ 2:1. Due to the high affinity, the Kd value of CYP105P1 was difficult to determine. The Kd value was estimated to be 0.66 ⫾ 0.18 ␮M for CYP105D6. Therefore, CYP105P1 showed higher binding affinity to filipin I than CYP105D6, exhibiting a good correlation with the catalytic activity. VOLUME 285 • NUMBER 22 • MAY 28, 2010

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FIGURE 4. Overall structures of CYP105P1-filipin I complex (A) and unliganded CYP105D6 (B), illustrated by ribbon representation. Heme and ligands are shown as stick models. The BC and FG loop regions are shown in dark gray. a.a., amino acids.

Crystal Structures of Filipin Hydroxylases

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formation in the H72A mutant structure (29) and is only slightly closed in the complex structure (Fig. 6B). Compared with the H72A mutant structure, the C␣ atoms of Asp-176 in the FG loop and Asp-75 in the BC loop shift by 8.7 and 2.6 Å, respectively. Root mean square deviations (r.m.s.d.) with previously determined structures are shown in supplemental Table S1. The long axis of the 28-membered filipin I ring was inclined at about 60 degrees from the vertical axis of the heme plane, and the pro-S hydrogen side of position C26 is directed toward the heme (Fig. 5). The distance between the C26 atom and heme iron is 5.0 Å, which appears to be appropriate for a monooxygenase reaction (discussed later). The filipin I molecule is surrounded by the heme, BC loop, FG loop, G helix, I helix, and C-terminal loop regions. The amino acid residues forming the pocket are as follows: Thr-79 — Pro-82 and Ser-86 —Trp-89 in the BC loop; Met-172—Met-173 in the FG loop; Thr-182—Glu-183, Gly-186 —Met-187, and Leu-189 — FIGURE 5. Interactions between CYP105P1 and filipin I. A, shown is an Fobs ⫺ Fcalc omit electron density map Gly-190 in the G helix; Met-228 — of the filipin I molecule contoured at 4.0 ␴. B, hydrophobic interactions are observed at both sides of the 28-membered ring. Labels for atoms of filipin I are underlined. C, a stereographic figure shows interactions with Asn-229, Gly-232—Thr-233, and the BC loop, FG helices, and I helix. The water molecules mediate hydrophilic interactions with the polyol group Ile-236 —Ala-238 in the I helix; Valof filipin I. The extensive hydrogen-bonding network and residues involved in it are shown as dotted lines and 388 —Phe-389 in the C-terminal stick models, respectively. The distance between the C26 atom of filipin I and the heme iron is 5.0 Å, and the loop (Fig. 5B). Among these, Gln-80 pro-S hydrogen side is directed toward the heme. and Pro-82 are the only two residues Structure of CYP105P1-Filipin I Complex—The crystal struc- to form direct hydrogen bonds with filipin I. The flat 28-memture of CYP105P1-filipin I complex was determined at 1.8 Å bered filipin I ring is sandwiched between two hydrophobic resolution and refined to an R factor of 18.8% (Rfree ⫽ 23.8%). faces. The ␤-face of the ring (see Rose et al. (40) for the definiThe crystal contains one molecule in the asymmetric unit and tion) is recognized by Pro-82, Leu-88, and Trp-89, and the exhibits a high Matthews coefficient (3.51 Å3/Da) and solvent ␣-face is recognized by Met-172, Met-173, Val-388, and Phecontent (65.0%). The final model contains residues from Asp-7 389. The C1 hydroxyl group of filipin I interacts with a carboxto His-403, including all four residues of the His tag: one heme, ylate moiety of heme through water-mediated hydrogen bonds. one filipin I molecule, 592 waters, and three sulfate ions. Fig. 4A The pocket at the polyol side of filipin I is filled with numerous shows the overall structure of CYP105P1. water molecules (Fig. 5C), and they mediate interactions with The electron density map for bound filipin I was clearly the FG loop, G helix, and BC loop regions. About 30 water observed in the distal pocket as shown in Fig. 5A. Superimpo- molecules are involved in this hydrogen-bonding network. The sitions with the ligand-free wild-type and H72A mutant struc- main chain atoms of Val-65, Val-77, Met-172, and Arg-174 and tures are shown in Fig. 6. The FG helices in the ligand-free the side chain atoms of Asp-75, Thr-79, and Thr182 are structures adopt an open conformation and close on substrate involved in the water-mediated recognition of the polyol side of binding. This region often adopts closing motion on ligand filipin I. In contrast, the pentaene side of filipin I forms hydrobinding (36 –39). The BC loop region consists of 33 amino acid phobic interactions with the I helix. Another important aspect residues and does not contain a helix, whereas most P450 struc- for substrate recognition is the K helix and subsequent loop tures have a B⬘ helix in this region (4). The BC loop region in the region (Fig. 7A). A pocket is formed between this region and the ligand-free wild-type structure has a unique conformation due heme, and the alkyl chain moiety of filipin I is bound at this to ligation of His-72 to the heme iron and completely covers the pocket. Three Gly residues, Gly-284, Gly-287, and Gly-288, are distal pocket (Fig. 6A), but this histidine ligation state is not clearly important to form this pocket. Moreover, in this region detectable in solution (29). The BC loop adopts an open con- there are several water-mediated hydrogen bonds with the C3

Crystal Structures of Filipin Hydroxylases

tion buffer. P450nor is closely related to the CYP105 family, but it catalyzes the reduction of nitric oxide to nitrous oxide using NADH as the direct electron donor (43). P450nor has an

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Fig. 8A shows superimposition of the active site structures of CYP105P1-filipin I and CYP105D6. The distances of C25, C26, C27, and C28 of filipin I from the heme iron of CYP105P1 is 6.7, VOLUME 285 • NUMBER 22 • MAY 28, 2010

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unusually large distal pocket even in the closed conformation in complex with an NADH analogue, nicotinic acid adenine dinucleotide (38). The distal pocket of P450nor is filled with numerous water molecules that form a proton channel, whereas most P450 enzymes have relatively tight hydrophobic pockets for their substrates. Structure of CYP105D6—The substrate-free crystal structure of CYP105D6 was determined at 2.3 Å resolution and refined to an R factor of 16.0% (Rfree ⫽ 22.1%). The crystal contains one molecule in the asymmetric unit and exhibits a normal Matthews coefficient (2.66 Å3/Da) and solvent content (53.9%). The final model contains residues from Ser-11 to His-408, including all four residues of the C-terminal His tag, one heme, and 277 waters. However, nine residues in the BC loop ranging from Arg-82 to Leu-90 and FIGURE 6. Closing motion of CYP105P1. Shown is a stereographic superimposition of the filipin I complex six residues in the FG loop ranging structure with the ligand-free wild-type (A) and H72A mutant (B) structures. BC loop and FG helices are colored from Gly-181 to Ala-186 were not magenta and green in ligand-free and complex structures, respectively. The filipin I molecule is shown as yellow included due to a disorder (Fig. 4B). sticks. In the ligand-free wild-type structure (A), the side chain of His-72 is ligated to the heme iron as the sixth In ligand-free P450 structures, ligand, and the BC loop sinks into the heme to completely cover the distal pocket. the BC loop region is relatively flexhydroxyl group of filipin I to stabilize substrate binding. The ible and sometimes disordered. For example, the ligand-free environment around the C1⬘ atom appears not to hinder the open structures of P450 PikC (14), P450 StaP (44), and CYP231A2 (45) have disordered BC loops. It is a notable feature binding of 1⬘-hydroxy filipin I. Comparison of the Substrate Binding Pocket with Other P450 of CYP105D6 that a total of 15 residues are disordered in both Enzymes—The volumes of substrate binding distal pockets the BC and FG loops, whereas the ligand-free CYP105P1 strucwere calculated using the complex structures of CYP105P1 and ture has only four disordered residues in the BC loop (29). The several P450s (Table 2). Supplemental Fig. S3 illustrates the overall structure of CYP105D6 is similar to those of ligand-free ligand binding pockets of CYP105P1, P450nor (CYP55A1), CYP105P1 structures. r.m.s.d. for 361 C␣ atoms was 2.4 Å with P450eryF, and P450cam (CYP101A). Among the P450s acting the ligand-free wild-type CYP105P1 structure, and r.m.s.d. upon macrolide substrates, P450 EryK has the largest pocket for 361 C␣ atoms was 2.1 Å with the H72A structure. The size, and CYP105P1 is the second largest. The pocket sizes basi- CYP105D6 structure shows relatively low structural similarcally correlate with substrate sizes. The substrate of P450 EryK ity to the structure of CYP105P1-filipin I complex (r.m.s.d. (erythromycin D) is far larger than that of P450eryF (6-deoxy- for 363 C␣ atoms ⫽ 2.5 Å), as the ligand-free CYP105D6 erythronolide B) due to insertion of two deoxysugar units structure is in an open state. Fig. 7B shows a superimposition (supplemental Fig. S1). The crystal structures of two CYP105 of CYP105D6 and CYP105P1-filipin I complex in the region family enzymes, P450 MoxA and P450 SU-1 (CYP105A1), have from the K helix to the ␤1–5 strand. It is clearly visible that been reported (41, 42). In contrast to the two position-specific CYP105D6 lacks a pocket for the alkyl side chain of filipin. filipin hydroxylases described in this study, both these enzymes The three glycine residues in CYP105P1 are replaced by can hydroxylate a wide variety of compounds. Their pocket bulky residues in CYP105D6, and a deletion of one residue sizes are completely different due to conformational differ- takes place in CYP105D6 (Fig. 7C). The side chains of Serences. The P450 SU-1 structure complexed with one of its sub- 290 and Ile-293 in CYP105D6 appear to hinder the binding strates, 1␣,25-dihydroxyvitamin D3, adopts a closed conforma- of the alkyl side chain, and thus, the C26 atom of filipin I tion. In contrast, the crystal structure of P450 MoxA adopts an cannot approach the heme iron. open conformation, although it binds a 2-morpholinoethanesulfonic acid (MES) molecule that is derived from a crystalliza- DISCUSSION

Crystal Structures of Filipin Hydroxylases

TABLE 2 Distal pocket volumes of P450 enzymes P450

Source organism

PDB code

Substrate/Liganda

Distal pocket volumeb Å3

CYP105P1 P450 EryK (CYP113A1) P450eryF (CYP107A1) P450epoK (CYP167A1) P450 PikC (CYP107L1) P450 SU-1 (CYP105A1) P450 MoxA (CYP105) P450nor (CYP55A1) CYP2B4 P450cam (CYP101A1) a b

S. avermitilis Saccharopolyspora erythraea S. erythraea Sorangium cellulosum Streptomyces venezuelae Streptomyces griseolus Nonomuraea recticatena Fusarium oxysporum Rabbit Pseudomonas putida

3ABA 2JJO 1JIO 1Q5D 2C7X 2ZBZ 2Z36 1XQD 1SUO 1DZ4

Filipin I (622.4) Erythromycin D (703.9) 6-Deoxyerythronolide B (386.5) Epothilone B (507.7) Narbomycin (509.7) 1␣,25-Dihydroxyvitamin D3 (416.6) MES (195.2) Nicotinic acid adenine dinucleotide (665.4) 4-(4-Chlorophenyl)imidazole (178.6) Camphor (152.2)

2166 2483 1247 1316 1860 1537 3285 3470 790 374

Values in parentheses are the molecular weights of the substrate or ligand. See supplemental Fig. S1 for the structures of substrates and ligands. Calculated by CASTp server with probe radius of 1.4 Å (50).

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FIGURE 7. Interactions between filipin I and a region from the K helix to ␤1–5 strand. A, a small pocket of CYP105P1 to accommodate the alkyl side chain is shown. This pocket is formed by a kink in a loop after the K helix that contains three glycine residues. Filipin I and water molecules are shown as green sticks and red spheres, respectively. B, superimposition of CYP105P1 (gray) and CYP106D6 (green) structures is shown. The side chains of Ser-290 and Ile-293 of CYP105D6 are shown by the dot surface of van der Waals radii. C, amino acid sequence alignment at a region from the K helix to subsequent strands is shown. Secondary structures of CYP105P1 are indicated above the sequence. Completely and relatively conserved regions are highlighted by black and white inverse characters and boxes, respectively. Residues labeled in panel B are underlined.

5.0, 5.3, and 4.5 Å, respectively. Superimposition with the ferrous dioxygen complex of P450cam suggests that the C26 atom is most appropriately positioned for a monooxygenase reaction, as C26 is more closely located to the C5 of camphor than C28 (Fig. 8B). Moreover, C26 of filipin I is located close to the O2 atoms of P450cam oxy-complex on the superimposition (about 3.1 Å to both oxygen atoms), but C28 is not (⬎3.4 Å). Thr-252 residue of P450cam is proposed to play an important role in protonation required for oxygen activation (46). The preceding acidic residue, Asp-251, is suggested to help proper positioning of Thr-252 and catalytic waters. The Asp/Glu-Thr pair is conserved in CYP105P1 (Asp-240 —Thr-241) and CYP105D6 (Glu-246 —Thr-247). In the case of P450eryF and CYP158A2, the Thr residue is replaced with Ala, but a hydroxyl group of their substrates substitutes for the Thr and helps to deliver the protons (47, 48). In the active site of CYP105P1, no hydroxyl group of the filipin I or 1⬘-hydroxyfilipin I substrate is positioned near the heme iron. Fig. 8C shows superposition with P450 EryK-erythromycin D. The target of hydroxylation site of erythromycin D (C12) is positioned 5.3 Å from the heme iron. A water molecule chain, which is thought to deliver proton from the bulk solvent to the active site, is present in P450cam and P450 EryK (Fig. 8, B and C) (13, 46). A conserved Glu residue (Glu-366 in P450cam and Glu-362 in P450 EryK) is involved in holding this water molecule chain. This Glu residue is also present in CYP105P1 (Glu-357) and CYP105D6 (Glu-362) and holds a water molecule in both structures (Fig. 8A). However, there are no water molecules near the active site of CYP105P1, as in the cases of P450eryF and P450 PikC (supplemental Fig. S4) (14, 47). In the substrate-free structure of CYP105D6, several water molecules are present near the heme iron (Fig. 8A). A water molecule is positioned 2.8 Å from the heme iron. These waters may be displaced on substrate binding, as type I spectral change is observed when filipin I is titrated (Fig. 3). In conclusion, a detailed catalytic mechanism of the filipin hydroxylases remains to be elucidated, but the general mechanism proposed for bacterial macrolide monooxygenases seems to be conserved. Compared with the previously reported structures (29), the CYP105P1-filipin I complex determined in this study provides clear structural insights into the mechanisms of substrate recognition. Filipin I is bound in a large pocket observed in the ligand-free H72A structure (29). The environment of the binding pocket specific for the shape and chemical nature of the

Crystal Structures of Filipin Hydroxylases

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nant in solution. A combination of crystallographic and kinetic analyses recently revealed that substrate binding by P450 EryK involved at least two steps because there was a pre-existing equilibrium between the open and closed subpopulations (13). There may also be a similar open-close equilibrium in the BC loop of CYP105P1. Two similar P450s catalyzing hydroxylations at different positions on the same substrate is an interesting feature. Filipin I is expected to bind to CYP105D6 in a “flipped” orientation relative to its binding with CYP105P1. However, the detailed mechanisms for CYP105D6 substrate recognition remain to be elucidated because we could not obtain a complex structure with the substrate. It is difficult to speculate on the possible binding mode of filipin I to this protein because the disordered regions are too long at the distal pocket. However, structural comparisons with the CYP105P1-filipin I complex revealed that filipin I cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation explains the strict regiospecificity of CYP105D6, which cannot catalyze hydroxylation of filipin I at position C26. The measurements of the catalytic activities against filipin I indicated that the 1⬘-hydroxylating activity of CYP105D6 was relatively less productive than the C26-hydroxylating activity of CYP105P1. FIGURE 8. Active site structures of CYP105P1-filipin I (green) superimposed with CYP105D6 (A, cyan), Moreover, spectral titration analysis P450cam-camphor-O2 (B, yellow), and P450 EryK-erythromycin D (C, magenta). Water molecules and the hydroxylation target positions of substrates (C26 of filipin I, C5 of camphor, and C12 of erythromycin indicated that the filipin I binding D) are shown as spheres. A water molecule is positioned 2.8 Å from the heme iron in the CYP105D6 to CYP105D6 was weaker than structure (A). CYP105P1. Although the production mechanism of filipin complex substrate explains the strict regio- and stereospecificity as well by S. filipinensis remains uncharacterized, our results probably as the efficient catalysis of 26S-hydroxylation by CYP105P1. explain why a natural filipin complex contains filipin II (1⬘The FG helices region adopts an open-close motion on sub- deoxyfilipin III), whereas 1⬘-hydroxyfilipin I is absent. When strate binding as similar to many other P450s, and this move- CYP105P1 and CYP105D6 were simultaneously incubated with ment appears to be sufficient for providing an entrance for the filipin I, 51.8, 20.9, 2.3, and 25.0% of filipin III, filipin II, 1⬘-hylarge substrate (see the supplemental movie). However, it is also droxyfilipin I, and filipin I were detected (data not shown). Filipossible that the filipin molecule enters through the region pin IV present in the filipin complex has been suggested to be a around the BC loop. This loop is thought to be highly flexible epimer of filipin III at C1⬘ or C3 (25). If filipin IV is the because it adopts distinct conformations among the three pre- 1⬘-epimer of filipin III, a possible CYP105D6 counterpart in viously reported structures (29). Moreover, spectroscopic anal- S. filipinensis likely to have ambiguity in its stereospecificity. ysis indicates that the His-ligated conformation of CYP105P1 The intrinsic flexibility of the alkyl side chain (26) may reduce in which the BC loop blocks substrate binding is not predomi- the stereospecificity of its hydroxylation reaction.

Crystal Structures of Filipin Hydroxylases Acknowledgments—We thank the staff of the Photon Factory for X-ray data collection and Dr. Jean-Marc Lancelin for providing the atomic coordinates of filipin III. REFERENCES

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1. Omura, S. (ed) (2002) Macrolide Antibiotics: Chemistry, Biology, and Practice, 2nd Ed., Academic Press, San Diego, CA 2. Xue, Y., and Sherman, D. H. (2001) Metab. Eng. 3, 15–26 3. Fjaervik, E., and Zotchev, S. B. (2005) Appl. Microbiol. Biotechnol. 67, 436 – 443 4. Ortiz de Montellano, P. R. (2005) Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd Ed., Kluwer Academic/Prenum Publishers, New York 5. Lamb, D. C., Waterman, M. R., Kelly, S. L., and Guengerich, F. P. (2007) Curr. Opin. Biotechnol. 18, 504 –512 6. Isin, E. M., and Guengerich, F. P. (2007) Biochim. Biophys. Acta 1770, 314 –329 7. McLean, K. J., Sabri, M., Marshall, K. R., Lawson, R. J., Lewis, D. G., Clift, D., Balding, P. R., Dunford, A. J., Warman, A. J., McVey, J. P., Quinn, A. M., Sutcliffe, M. J., Scrutton, N. S., and Munro, A. W. (2005) Biochem. Soc. Trans. 33, 796 – 801 8. Munro, A. W., Girvan, H. M., and McLean, K. J. (2007) Nat. Prod. Rep. 24, 585– 609 9. Poulos, T. L. (2007) Drug Metab. Rev. 39, 557–566 10. Guengerich, F. P. (2002) Nat. Rev. Drug Discov. 1, 359 –366 11. Cupp-Vickery, J. R., and Poulos, T. L. (1995) Nat. Struct. Biol. 2, 144 –153 12. Nagano, S., Li, H., Shimizu, H., Nishida, C., Ogura, H., Ortiz de Montellano, P. R., and Poulos, T. L. (2003) J. Biol. Chem. 278, 44886 – 44893 13. Savino, C., Montemiglio, L. C., Sciara, G., Miele, A. E., Kendrew, S. G., Jemth, P., Gianni, S., and Vallone, B. (2009) J. Biol. Chem. 284, 29170 –29179 14. Sherman, D. H., Li, S., Yermalitskaya, L. V., Kim, Y., Smith, J. A., Waterman, M. R., and Podust, L. M. (2006) J. Biol. Chem. 281, 26289 –26297 15. Wachtler, V., and Balasubramanian, M. K. (2006) Trends Cell Biol. 16, 1– 4 16. Gimpl, G., and Gehrig-Burger, K. (2007) Biosci. Rep. 27, 335–358 17. Butler, J. D., Comly, M. E., Kruth, H. S., Vanier, M., Filling-Katz, M., Fink, J., Barton, N., Weintroub, H., Quirk, J. M., and Tokoro, T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 556 –560 18. Butler, J. D., Blanchette-Mackie, J., Goldin, E., O’Neill, R. R., Carstea, G., Roff, C. F., Patterson, M. C., Patel, S., Comly, M. E., and Cooney, A. (1992) J. Biol. Chem. 267, 23797–23805 19. Whitfield, G. B., Brock, T. D., Ammann, A., Gottlieb, D., and Carter, H. E. (1955) J. Am. Chem. Soc. 77, 4799 – 4801 20. Bergy, M. E., and Eble, T. E. (1968) Biochemistry 7, 653– 659 21. Rychnovsky, S. D., and Richardson, T. I. (1995) Angew. Chem. Int. Ed. Engl. 34, 1227–1230 22. Richardson, T. I., and Rychnovsky, S. D. (1996) J. Org. Chem. 61, 4219 – 4231 23. Pandey, R. C., and Rinehart, K. L., Jr. (1970) J. Antibiot. 23, 414 – 417

24. Edwards, D. M. F. (1989) J. Antibiot. 42, 322–324 25. Pandey, R. C., Narasimhachari, N., Rinehart, K. L., Jr., and Millington, D. S. (1972) J. Am. Chem. Soc. 94, 4306 – 4310 26. Volpon, L., and Lancelin, J. (2000) FEBS Lett. 478, 137–140 27. Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, Y., Hattori, M., and Omura, S. (2003) Nat. Biotechnol. 21, 526 –531 28. Chen, L., Chen, J., Jiang, Y., Zhang, W., Jiang, W., and Lu, Y. (2009) FEMS Microbiol Lett. 298, 199 –207 29. Xu, L. H., Fushinobu, S., Ikeda, H., Wakagi, T., and Shoun, H. (2009) J. Bacteriol. 191, 1211–1219 30. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307–326 31. Vagin, A., and Teplyakov, A. (1997) J. Appl. Cryst. 30, 1022–1025 32. Emsley, P., and Cowtan, K. (2004) Acta Crystallogr. D Biol. Crystallogr. 60, 2126 –2132 33. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. D. Biol. Crystallogr. 53, 240 –255 34. Schu¨ttelkopf, A. W., and van Aalten, D. M. (2004) Acta Crystallogr. D. Biol. Crystallogr. 60, 1355–1363 35. Painter, J., and Merritt, E. A. (2006) J. Appl. Crystallogr. 39, 109 –111 36. Park, S. Y., Yamane, K., Adachi, S., Shiro, Y., Weiss, K. E., Maves, S. A., and Sligar, S. G. (2002) J. Inorg. Biochem. 91, 491–501 37. Poulos, T. L. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 13121–13122 38. Oshima, R., Fushinobu, S., Su, F., Zhang, L., Takaya, N., and Shoun, H. (2004) J. Mol. Biol. 342, 207–217 39. Zhao, B., Guengerich, F. P., Bellamine, A., Lamb, D. C., Izumikawa, M., Lei, L., Podust, L. M., Sundaramoorthy, M., Kalaitzis, J. A., Reddy, L. M., Kelly, S. L., Moore, B. S., Stec, D., Voehler, M., Falck, J. R., Shimada, T., and Waterman, M. R. (2005) J. Biol. Chem. 280, 11599 –11607 40. Rose, I. A., Hanson, K. R., Wilkinson, K. D., and Wimmer, M. J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2439 –2441 41. Yasutake, Y., Imoto, N., Fujii, Y., Fujii, T., Arisawa, A., and Tamura, T. (2007) Biochem. Biophys. Res. Commun. 361, 876 – 882 42. Sugimoto, H., Shinkyo, R., Hayashi, K., Yoneda, S., Yamada, M., Kamakura, M., Ikushiro, S., Shiro, Y., and Sakaki, T. (2008) Biochemistry 47, 4017– 4027 43. Nakahara, K., Tanimoto, T., Hatano, K., Usuda, K., and Shoun, H. (1993) J. Biol. Chem. 268, 8350 – 8355 44. Makino, M., Sugimoto, H., Shiro, Y., Asamizu, S., Onaka, H., and Nagano, S. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 11591–11596 45. Ho, W. W., Li, H., Nishida, C. R., de Montellano, P. R., and Poulos, T. L. (2008) Biochemistry 47, 2071–2079 46. Nagano, S., and Poulos, T. L. (2005) J. Biol. Chem. 280, 31659 –31663 47. Nagano, S., Cupp-Vickery, J. R., and Poulos, T. L. (2005) J. Biol. Chem. 280, 22102–22107 48. Zhao, B., Guengerich, F. P., Voehler, M., and Waterman, M. R. (2005) J. Biol. Chem. 280, 42188 – 42197 49. Lovell, S. C., Davis, I. W., Arendall, W. B., 3rd, de Bakker, P. I., Word, J. M., Prisant, M. G., Richardson, J. S., and Richardson, D. C. (2003) Proteins 50, 437– 450 50. Dundas, J., Ouyang, Z., Tseng, J., Binkowski, A., Turpaz, Y., and Liang, J. (2006) Nucleic Acids Res. 34, W116 –W118

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